The long-term goal of our research is to elucidate the mechanism of eukaryotic DNA replication and its control. Growing cells must duplicate their genetic information with a high degree of accuracy and do so once and only once per cell cycle. Alterations in the cell that lead to changes in these requirements result in cell death or abnormal proliferation that can result in cancer. Many of the details involved in replication and its control remain poorly understood. Our plans are to examine the function of critical replication proteins that interact with origins and in a step-wise manner lead to the initiation and elongation of DNA chains. For this purpose, we propose to study: a) the interaction of the origin recognition complex with DNA and its reactions with a number of protein that result in the origin-loading of the mini-chromosome maintenance (Mcm) protein complex; b) the interaction of the pre-replication complex with key proteins that activate the Mcm complex which is thought to act as the replicative helicase at the replication fork. Events critical for this activation include modification of the Mcm complex by the action of Cdc7-Dbf4 and a cyclin-dependent kinase as well as the Mcml0p-dependent recruitment of Cdc45. c) We plan to investigate the multiple roles of Mcml0p which activate the Cdc7-Dbf4 kinase catalyzed phosphorylation of the Mcm complex and the action of DNA polymerase alpha-primase complex, d) We will characterize the properties of the four subunit DNA polymerase epsilon complex which we have cloned. This polymerase plays critical roles in the formation of the replication machinery at the fork and is reported to participate in events prior to initiation of DNA synthesis; e) to examine two recently identified clamp loader derivatives of RFC:Rad17-RFC involved in checkpoint regulation of replication in conjunction with the clamp complex Rad9-Rad1-Hus1 and Ctf18, which complexed to Dcc1, Ctf8 and RFC2-5 subunits, is involved in sister-chromatid pairing.